The paraventricular nucleus of the hypothalamus (PVH) is a pivotal structure in organizing adaptive responses to emotional stress, largely by virtue of its indispensable role in activating pituitary-adrenal (glucocorticoid) responses. Four sets of experiments are proposed to clarify the circuits and cellular mechanisms that mediate PVH responses to acute emotional stress, and how these come to be modified by repeated stress exposure. First, an immediate-early gene-guided ablation strategy will test the hypothesis that PVH responses to an acute emotional stress, footshock, are mediated via spinal nociceptive pathways that access the forebrain via the thalamus. A classical conditioning paradigm will be used to determine whether and how the afferent mediation of PVH recruitment by acute footshock may be transferred to pathways subserving a different sensory modality. Second, we will test the hypothesis that catecholamine inputs modulate PVH responses to acute emotional stress by reinforcing a primary drive initiated by other systems. The functional organization of local inhibitory inputs to PVH will be characterized by assessing stress and steroidal influences on identified sources of GABAergic afferents. We will attempt to establish a method for selective GABAergic denervation of PVH, to allow the role of this innervation to be assessed directly. Immunotoxin ablations will be used to [pursue] preliminary indications of an involvement of the orexin peptide system in emotional stress effects on PVH. Third, transcriptional profiling of microdissected PVH subregions will be carried out to test the hypothesis that intrinsic mechanisms are involved in habituation of PVH response to repeated emotional stress. Animals with controlled glucocorticoid levels will be used to determine how and where the hormone interacts with afferent circuitry to effect habituation. Finally, to begin to relate circuitry to cellular mechanisms, pharmacologic methods will be used to assess the role of adrenergic neurotransmission in acute stress effects on the transcription of genes encoding peptides that interact to govern pituitary-adrenal output. Lentiviral vector-mediated gene transfer will be used to evaluate the role in this context of a key transcription factor, the cyclic AMP response element-binding protein. Glucocorticoid products of the pituitary-adrenal system facilitate coping with acute emergencies, but sustained elevations in hormone levels, as seen in chronic emotional stress, is a contributing or exacerbating factor in diverse systemic, psychiatric and even neurodegenerative disorders. A fuller basic understanding of circuits and mechanisms underlying these adaptations will promote more effective management of stress related disease.